The importance of specific citrullinated clusterin and vimentin found in a multi-coloured bead-based citrulline-peptide array system in rheumatoid arthritis

Objective The importance of citrullination in rheumatoid arthritis (RA) has been reported, but the degree to which individual citrullinated proteins affect the onset and progression of RA is still unclear. We aimed to identify citrullinated proteins that may play an important role in the onset and progression of RA using an individualised anti-citrullinated protein antibody (ACPA) evaluation system with citrullinated peptides as probes. Methods Serum samples from 50 normal donors and 51 RA patients were evaluated using a custom MagPlexTM bead array with 13 types of citrullinated peptide. The presence/absence of ACPAs that react to each citrullinated peptide in each subject was determined using the Z-score, which was calculated based on the fluorescence intensity distribution of a sample from a normal donor. Whether the fluorescence intensity was inhibited when free citrullinated peptides were added to a system was also evaluated. Results Median fluorescence intensities obtained from beads coupled with the 13 types of citrullinated peptide were all significantly higher in RA patients versus normal donors. With a Z-score >= 2 as the cut-off value for the presence of ACPAs, ACPAs that recognised five types of citrullinated peptides derived from fibrinogen A, fillagrin, clusterin, and vimentin were widely detected in RA patients. In addition, inhibition experiments showed that citrullinated vimentin, clusterin, and enolase 1A peptides inhibited coupling of ACPAs to other citrullinated peptides. Conclusion ACPAs to many citrullinated proteins exhibited cross-reactivity to citrullinated clusterin and vimentin, suggesting the importance of citrullinated clusterin and vimentin in the early stages of RA pathogenesis.